Download PureLink™96 HQ Mini Plasmid Purification Kit

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A P P L I CAT I O N B IOPHARM A
PureLink 96
™
PureLink 96 HQ Mini
Plasmid Purification Kit
™
Automated, high throughput purification of plasmid DNA
The ever-increasing demand for high
throughput (HTP) applications for
molecular techniques, particularly
plasmid DNA purification methods,
has led to a number of new automated
purification procedures in life sciences
laboratories. To meet the needs of these
automated methods, Invitrogen offers
you the PureLink™96 HQ (High Quality)
Mini Plasmid Purification Kit. This kit
provides a universal 96-well plate design
that not only is compatible with most
automated liquid handling platforms
but also has the ability to deliver the
high quality plasmid DNA necessary for
today’s stringent applications. Use the
PureLink™96 HQ Mini Plasmid Purification
Kit with your robotic system to quickly
generate high mini-prep yields with low
genomic DNA contamination and nondetectable RNA contamination, without
compromising quality.
Workhorse for plasmid prep purification
The PureLink™96 HQ Mini Plasmid
Purification Kit is based on an alkaline /
SDS cell lysis procedure, followed by
neutralization of the lysate and selective
plasmid DNA binding to glass microfiber
filters. The bound DNA is then washed
to remove impurities from the original
sample (such as genomic DNA and RNA),
and the clean DNA is eluted in a lowsalt buffer. This DNA isolation method is
typically faster and easier to perform than
anion exchange methods. There’s no need
Figure 1:
1. Wash station 2. Disposable tip (DiTi) waste 3. Disposable tip carrier 4. Wash Buffer
5. Elution Buffer 6. Resuspension Buffer 7. Lysis Buffer 8. Neutralization/Binding Buffer
9. Te-Shake shaker with deep-well plate
Tecan Journal 1/2007
for alcohol precipitation or desalting,
making it ideal for HTP studies.
Validated HTP procedure using Tecan
automated robot
To get you started on your HTP
applications, we have validated a robust
procedure to isolate plasmid DNA from
bacteria in a 96-well format using the
PureLink™96 HQ Mini Plasmid
Purification Kit and a Tecan Freedom
EVO® automated liquid handling platform
with Freedom EVOware® software.
The Freedom EVO separation system
with the Te-VacS™ vacuum separation
module allows for walkaway automated
processing. Pipetting is performed by
the liquid handling (LiHa) arm, while
the robotic manipulator (RoMa) arm
performs all transports of the plates and
assembling steps of the Te-VacS vacuum
manifold.
Briefly, the PureLink™96 HQ Mini Plasmid
Purification Kit script opens with the
deck layout as shown in Figures 1 and 2.
The pelleted cell culture plate is placed
on the Te-Shake™ orbital mixer. Cell
resuspension, lysis, and neutralization
buffers are added sequentially, with
shaking between each buffer addition.
The binding and clarification plates are
APPLICATIONS B IOPHA R M A
Figure 2: The Freedom EVO workstation layout
The filter is then washed with buffer W1
before addition of elution buffer. This
script allows for anywhere from 1 to 96
samples to be processed at a time, with
up to 1.5 × 109 cells per well in <1 hour per
96-well plate.
Figure 3: High quality plasmid DNA is
obtained using the Purelink™96 HQ Mini
Plasmid Purification Kit
Figure 4: The Freedom EVO provides uniformity
and consistency in DNA yield and quality
deployed to the Te-VacS using the RoMa.
The neutralized lysate is transferred
to the clarification plate, whereupon
the vacuum is applied. The cleared
lysate is collected in the binding plate
and transferred to the second Te-VacS
manifold and the vacuum applied to bind
the plasmid DNA to the glass fiber matrix.
High quality, uniform and
consistent results
Plasmid DNA extracted from the
PureLink™96 HQ Mini Plasmid Purification
Kit via the Tecan robot is of extremely
high quality as evidenced by the absence
of RNA, extremely low genomic DNA
contamination, and high supercoiled to
nicked form ratios (Figure 3). In addition,
uniformity and consistency in DNA yield
and quality among the different wells
indicate that the Freedom EVO separation
system provides a very reproducible
method of automated plasmid DNA
isolation (Figure 4). Well-to-well variation
was on average <10% for the automated
protocol. The resulting plasmid DNA is
transfection quality, with low endotoxin
levels, and suitable for use in fluorescent
sequencing with >98% accuracy of base
identity for a 600-base read.
“
The Freedom EVO
platform with the
Te-VacS vacuum
separation module
allows walkaway
automated
processing
”
Efficient, quick, reliable and easily
automated HTP method
The PureLink™96 HQ Mini Plasmid
Purification Kit provides an efficient,
quick, reliable, and easily automated
HTP method for plasmid DNA sample
preparation. Similar scripts have also
been written for the PureLink™96 PCR
Purification Kit and PureLink™96 RNA
Purification Kit on Freedom EVOware
software.
This article is reprinted with permission
from Invitrogen. For more infromation,
visit www.invitrogen.com/purelink
PureLink is a trademark of Invitrogen
Tecan Journal 1/2007
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